In general, human insulin precursor ("proinsulin") has been prepared in the course of manufacturing mature insulin ("insulin") by the recombinant DNA technology which comprises a step of inserting a structural gene into a plasmid DNA of E. coli.
As shown in FIG. 1, a fusion protein containing the proinsulin is expressed in the form of inclusion body in E. coli, and the inclusion bodies obtained by centrifugation after lysis of the cells are washed with non-ionic or ionic detergent, or with a denaturant at a low concentration. Such a treatment accompanied by centrifugation is repeated to result in increase of purity of the desired protein (see: Mukhopadhyay, A. et al., Advances in Biochemical Engineering/Biotechnology, 56, 61-108, 1997). In order to minimize intermolecular hydrophobic interaction and formation of incorrect disulfide bonds, the washed inclusion bodies are dissolved in a denaturant such as urea or guanidine.HCl solution containing a reducing agent such a dithiothreitol (DTT) or 2-mercaptoethanol, or in NaOH (see: Fischer et al., Biotechnology & Bioengineering, 41, 3-13, 1993). The dissolved inclusion bodies are centrifuged at a high speed, and the supernatant is diluted with cold water to recover the inclusion bodies as a precipitate (see: EP 0 055 945 A2). The inclusion bodies thus obtained contain a fusion protein of proinsulin and a heteroprotein such as .beta.-galactosidase, which are linked by a cross-linkage of methionine residue. The fusion protein is treated with cyanogen bromide (CNBr), and substitution of six (6) --SH groups present in proinsulin with --SSO.sub.3 groups follows to give proinsulin S-sulfonate. Such a sulfonation step leads to increase in stability of insulin precursor and efficiency of a later refolding reaction (see: EP 0 055 945 A2). The proinsulin S-sulfonate is refolded to have a native conformation by using reducing agents such as 2-mercaptoethanol, DTT, etc., or a redox system such as glutathione (see: Fischer et al., Biotechnology & Bioengineering, 43, 3-13, 1993). The native proinsulin thus obtained is converted into biologically active insulin by removing X (or C chain) which links A chain and B chain through the treatment of trypsin and carboxypeptidase B (see: Kemmler W., et al., J.B.C., 246, 6786-6790, 1971). Finally, insulin is purified through a reverse-phase high performance liquid chromatography (RP-HPLC), etc. (see: Kroeff, E. P., et al., J Chromatogr., 461, 45-61, 1989) and crystallized by the technique of Zn-crystallization (see: Mirsky, et al., J. Clinical Investigation, 42, 1869-1872, 1963; Brader, M. L., TIBS, 16, 341-345, 1991).
The conventional process for preparing proinsulin or insulin is, however, proven to be less satisfactory in the senses that: it accompanies complicated steps of dissolution and sulfonation, purification, concentration and desalting; and it employs an inefficient refolding reaction, which results in decreased yield of the desired protein. Accordingly, there are strong reasons for exploring and developing an improved process for preparing proinsulin or insulin in a simple and efficient manner, while preserving its biological activity.